Breast cancer is the most common malignancy among females in most western countries, who have an overall lifetime risk of more than 10 percent for developing invasive breast cancer (Feuer, et al., J Natl Cancer Inst, 85:892-897, 1993). Early detection is crucial to successful treatment, and various approaches are being developed in the art for breast cancer diagnosis and/or for determining responsiveness to treatment. Such approaches include: determination of hormone receptor status (e.g., positivity); gene expression profiling (mRNA/cDNA); and DNA methylation marker characterization.
Hormone receptor status. Clinical and epidemiological studies have previously suggested that breast cancer is comprised of at least two distinct groups based on hormone receptor status (Potter, et al., Cancer Epidemiol Biomarkers Prev, 4:319-326, 1995; Fox, M. S., Jama, 241:489-494, 1979). The presence of estrogen receptors (ER) and/or progesterone receptors (PR) is an important diagnostic feature of breast cancer, both reflective of disease etiology (Potter, et al., Cancer Epidemiol Biomarkers Prev, 4:319-326, 1995), and predictive of response to treatment with the antiestrogen tamoxifen (Anonymous, Lancet, 351:1451-1467, 1998; Bardou, et al., J Clin Oncol, 21:1973-1979, 2003).
However, even with knowledge of hormone receptor status, considerable controversy currently exists as to which breast cancer patients will benefit from antiestrogen (e.g., tamoxifen) treatment. Antiestrogens primarily function through their ability to compete with available estrogens for binding to ER. However, the consequences of occupying, for example, ER with an antiestrogen appear dependent upon the cellular context, which ER is occupied (ER α and/or ER β), and perhaps the structure of the ligand (Clarke et al., Phamacological Reviews, 53:25-72, 2001; Cellular and Molecular Pharmacology of Antiestrogen Action and Resistance).
Expression profiling. More recently, molecular profiling of breast cancer using gene expression (cDNA) microarrays to determine gene expression profiles has led to a further refinement of the subclassification of breast cancer into five major distinct subtypes/clusters, comprised of one basal-like, one ERBB2-overexpressing, two luminal-like, and one normal breast tissue-like subgroup (Sorlie, T., et al., Proc Natl Acad Sci USA, 100:8418-8423, 2003). Such expression profiling studies have also led to the identification of gene expression signatures associated with prognosis (Perou, et al., Nature, 406:747-752, 2000, Sorlie, et al., Proc Natl Acad Sci USA, 98:10869-10874, 20015-9).
Molecular profiling in breast cancer has, however, thus far focused primarily on the use of cDNA microarrays, which are limited by the innate instability of RNA and are poorly compatible with formalin fixation and parafilm embedding of tumor tissues, typically used in routine histopathology.
DNA methylation/Gene silencing. Significant progress has been made in recent years towards the implementation of DNA methylation markers as clinical tools in cancer detection and diagnosis, and DNA methylation markers provide an alternative approach to molecular profiling (Laird, P. W., Nat Rev Cancer, 3:253-266, 2003). Hypermethylation of promoter CpG islands, frequently observed in breast cancer (Yan, P. S., et al., Cancer Res, 61:8375-8380, 2001, Yang, X., et al., Endocr Relat Cancer, 8:115-127, 2001, Widschwendter & Jones, Oncogene, 21:5462-5482, 2002), is often associated with transcriptional silencing of the associated gene, thus providing a DNA-based surrogate marker for expression status (Jones & Baylin, Nat Rev Genet, 3:415-428, 2002).
Microarray-based methods of DNA methylation analysis are, however, hampered by modest quantitative accuracy, poor sensitivity to low levels of CpG island hypermethylation and technical challenges in target DNA preparation, which requires either bisulfite-PCR-amplification of each individual locus (Adorjan, et al., Nucleic Acids Res, 30:E21, 2002), or the use of restriction enzyme digestion (Yan, P. S., et al., Cancer Res, 61:8375-8380, 2001), which is not consistently reliable with formalin-fixed tissues.
Tamoxifen. Tamoxifen was introduced over 25 years ago, and has been the mainstay of the endocrine adjuvant treatment of breast cancer. Tamoxifen has become the most widely used anticancer drug, and may be considered one of the first targeted therapies (Jordan, V. C., Nat Rev Drug Discov, 2:205-213, 2003). Tamoxifen, an ‘anti-estrogen,’ is a selective estrogen-receptor modulator, and has been shown to dramatically reduce the risk of breast cancer (Powles, T. J., Nat Rev Cancer, 2:787-794, 2002) and of breast cancer recurrence (Jordan, V. C., Nat Rev Drug Discov, 2:205-213, 2003). TAM is a classical partial agonist and exhibits both species and tissues specificity for inducing either an agonist or antagonist response. In rats and humans, it exhibits partial agonism (e.g., producing antagonist effects in the breast, but agonist effects in the vagina and endometrium. Long-term TAM use is generally associated with a reduced incidence of contralateral breast cancer (antagonist), a reduced incidence of primary breast cancer in high-risk women (antagonist), maintenance of bone density (agonist), and increased risk of endometrial carcinomas (agonist). Other antiestrogens are known in the art, and some of these also act through the ER.
Breast Cancer Treatment. Assessment of a patient's condition relative to defined classifications of the disease is typically the first step of any breast cancer treatment, and the value of such assessment is inherently dependent upon the quality of the classification. Breast cancers are staged according to size, location and occurrence of metastasis. Methods of treatment include the use of surgery, but additionally include radiation therapy, chemotherapy and endocrine therapy, which are also used as adjuvant therapies to surgery. Generally, more aggressive diseases are regarded as requiring treatment with more aggressive therapies.
Although the vast majority of early cancers are operable, (i.e., the tumor can be completely removed by surgery), about one third of the patients with lymph-node negative diseases and about 50-60% of patients with node-positive disease will develop metastases during follow-up. Based on this observation, systemic adjuvant treatment has been introduced for both node-positive and node-negative breast cancers. Systemic adjuvant therapy is administered after surgical removal of the tumor, and has been shown to reduce the risk of recurrence significantly. Several types of adjuvant treatment are available, including, but not limited to: endocrine treatment, also called hormone treatment (for hormone receptor positive tumors); different chemotherapy regimens; and antibody and antibody-based treatments, based on novel agents like Herceptin™ (HER-2-specific antibody).
The growth of the majority (about 70-80%) of breast cancers is dependent on the presence of estrogen. Therefore, one important target for adjuvant therapy is the removal of estrogen (e.g., by ovarian ablation), the blocking of its synthesis or the blocking of its actions on the tumor cells, either by blocking the receptor with competing substances (e.g., Tamoxifen) or by inhibiting the conversion of androgen into estrogen (e.g., aromatase inhibitors). This type of treatment is referred to in the art as ‘endocrine treatment.’ Endocrine treatment is thought to be efficient only in tumors that express hormone receptors (the estrogen receptor (ER), and/or the progesterone receptor (PR)). Currently, the vast majority of women with hormone receptor positive (HR+) breast cancer receive some form of endocrine treatment, independent of their nodal status. The most frequently used drug in this scenario is Tamoxifen.
However, even in hormone receptor positive patients, not all patients benefit from endocrine treatment. Adjuvant endocrine therapy reduces mortality rates by 22% while response rates to endocrine treatment in the metastatic (advanced) setting are 50 to 60%.
Because Tamoxifen has relatively few side effects, treatment may be justified even for patients with low likelihood of benefit. However, these patients may require additional, more aggressive adjuvant treatment. Even in earliest and least aggressive tumors, such as node-negative, hormone receptor positive tumors, about 21% of patients relapse within 10 years after initial diagnosis if they receive Tamoxifen monotherapy as the only adjuvant treatment (Lancet. 351:1451-67, 1998; Tamoxifen for early breast cancer: an overview of the randomized trials; Early Breast Cancer Trialists' Collaborative Group). Similarly, some patients with hormone receptor negative disease may be treated sufficiently with surgery and potentially radiotherapy alone, whereas others may require additional chemotherapy.
Several cytotoxic regimens have shown to be effective in reducing the risk of relapse in breast cancer. According to current treatment guidelines, most node-positive patients receive adjuvant chemotherapy both in the US and Europe, because the risk of relapse is considerable. Nevertheless, not all patients do relapse, and there is a proportion of patients who would never have relapsed even without chemotherapy, but who nevertheless receive chemotherapy due to the currently used criteria. In hormone receptor positive patients, chemotherapy is usually given before endocrine treatment, whereas hormone receptor negative patients receive only chemotherapy.
The situation for node-negative patients is particularly complex. In the US, cytotoxic chemotherapy is recommended for node-negative patients, if the tumor is larger than 1 cm. In Europe, chemotherapy is considered for the node-negative cases if one or more risk factors is present, such as: tumor size larger than 2 cm; negative hormone receptor status; tumor grading of three; or age <35. Generally, there is a tendency to select premenopausal women for additional chemotherapy whereas for postmenopausal women, chemotherapy is often omitted. Compared to endocrine treatment, in particular that with Tamoxifen or aromatase inhibitors, chemotherapy is highly toxic, with short-term side effects such as nausea, vomiting, bone marrow depression, as well as long-term effect, such as cardiotoxicity and an increased risk for secondary cancers.
Tamoxifen treatment responsiveness. Hormone receptor (HR) status, defined as either ER and/or PR positivity, has been shown to predict response to tamoxifen treatment (Anonymous, Lancet, 351:1451-1467, 1998, Bardou, et al., J Clin Oncol, 21:1973-1979, 2003). Interestingly, although tamoxifen is thought to act through the ER, PR status is an independent factor predictive of adjuvant endocrine treatment benefit (Bardou, et al., J Clin Oncol, 21:1973-1979, 2003).
Need for improved diagnostic and prognostic assays. While the individual approaches described above (hormone receptor status, gene expression profiling, and DNA methylation markers) have provided real benefit in cancer treatment, an even greater benefit could be attained through a better understanding of possible interactions between DNA methylation and hormone receptor biology.
There is, therefore, a pronounced need in the art to investigate and characterize the degree of interaction between DNA methylation and hormone receptor biology in cancer, and particularly in breast cancer. There is a pronounced need in the art for novel methods for determining hormone receptor (e.g., ER and PR) status in cancers, particularly breast cancer.
There is a pronounced need in the art for novel prognostic methods indicative of disease progression and/or survival in breast cancer patients. There is a pronounced need in the art for novel predictive methods indicative of clinical response to therapy in breast cancer patients (e.g., patients treated with endocrine (e.g., tamoxifen) therapy). There is a pronounced need in the art for novel methods that are both prognostic and predictive of response in and non-treated and tamoxifen-treated breast cancer patients. There is a need for novel methods allow for better selection of patients for chemotherapy or other, more aggressive forms of breast cancer therapy.